Increased Efficiency of Biomolecule Identification by Optimization of Trypsin Digestion Buffers

Transcription

1 Increased Efficiency of Biomolecule Identification by Optimization of Trypsin Digestion Buffers Phillip Humphryes, Valeria Barattini; Thermo Fisher Scientific, Runcorn, UK Application Note 274 Key Words SMART Digest, peptides, monoclonal antibody, protein, proteomics, trypsin digestion, buffer, mab, biomolecules, biotherapeutics, biopharmaceutical Goal To demonstrate how the Thermo Scientific SMART Digest kit removes the uncertainty associated with conventional solution-based tryptic digestion protocols, resulting in higher efficiency and sample characterization. Introduction Protein insolubility can be a major impediment to traditional bottom-up proteomic methods becauses it reduces the efficiency of enzymatic digestions and makes analysis problematic. Insolubility can be caused by a variety of factors, including the salt concentration, the presence of various organic solvents, temperature, or simply the nature of the protein itself. In some cases, this insolubility can be useful for purification purposes as is seen with immunoprecipitation or pre-pelleting protocols. However, in most cases, precipitation of the protein out of solution is undesirable. The digestion itself can increase the solubility of a protein as the resulting peptides, due to their smaller size, are more soluble than their parent proteins or corresponding polypeptide chains. Protein solubility can be enhanced by the addition of small amounts of solvents, detergents, and/ or chaotropes; however, many of these methods are also detrimental to trypsin digestion. The SMART Digest buffer was developed to maintain protein solubility while increasing the rate of digestion. This was achieved by optimizing the solubility buffer then the digestion buffer.

2 2 Experimental Digestion SMART Digest Kit (P/N 69-) Chemicals Fisher BioReagents bovine serum albumin (BSA) (P/N 28563) Thermo Scientific Pierce trypsin protease (P/N ) Fisher BioReagents Tris buffered saline (TBS) (P/N ) Fisher Scientific Optima LC-MS water (P/N 9564) Fisher Scientific Optima acetonitrile (ACN) (P/N 334) Fisher Scientific Optima isopranol (IPA) (P/N 934) Fisher Scientific dimethyl sulfoxide (DMSO) (P/N 55) Fisher Scientific Optima methanol (MeOH) (P/N 394) Fisher BioReagents 2,2,2-trifluoroethanol (TFE) (P/N ) Thermo Scientific Pierce guanidine HCl (P/N 82365) Fisher BioReagents Tween 2 (P/N 33) Thermo Scientific Pierce octyl glucoside (P/N 283) Fisher BioReagents deoxycholate (P/N ) Sample Handling Equipment Heater/shaker equipped with PCR block and heated lid Experiment : Solubility buffer comparison Bovine serum albumin (BSA) was dissolved in the following buffers: Tris buffered saline (TBS) TBS with trypsin (:5 trypsin to protein ratio) An optimized proprietary solubility buffer Final concentrations of BSA of.25,,., and mg/ml were produced. Experiment 2: Digestion buffer comparison A mg/ml solution of BSA in the optimized digestion buffer was prepared and compared to BSA in optimized digestion buffer with the organic solvents, chaotropes, surfactants, and detergent additives listed in Table. Samples were then digested using the SMART Digest kit at 4 rpm and 7 ºC using a range of different incubation times (3, 6, 9, and 2 minutes). Table. List of additives and concentrations used to assess BSA solubility. Additives Concentrations ACN, 5,, 2% IPA, 5,, 2% DMSO, 5,, 2% MeOH, 5,, 2% TFE, 5,, 2% Guanidine HCl.5M Tween 2.5%,.5% Octylglucoside Deoxycholate

3 Results and Discussion Experiment One: Solubility Buffer Comparison Data in Figure shows BSA solubility up to.25 mg/ml in the TBS solution, which further increased to mg/ml with the addition of trypsin (Figure 2) for the 5 min time points. This increase in solubility on addition of trypsin can largely be attributed to the effect of digestion of the protein to its constituent peptides, which are generally more soluble than the parent protein Figure. BSA solubility in TBS solution. Concentrations of BSA greater than mg/ml in TBS/ trypsin solutions (Figure 2) displayed precipitation after only minutes of incubation. This can be attributed to the denaturation of the trypsin, due to the high temperature, limiting the extent to which digestion of the protein could be achieved. Figure 2. BSA solubility in TBS solution with trypsin. By contrast, the optimized solubility buffer was capable of solubilizing all concentrations of BSA up to as high as mg/ml (Figure 3). This is five times higher than the other approaches Figure 3. BSA solubility in optimized buffer.

4 4 Experiment Two: Digestion Buffer Comparison The solubility of BSA in digestion buffer samples was assessed after digestion with the SMART Digest kit (Figure 4). The digestion buffer showed excellent solubility with incubation times ranging from 3 to 2 minutes ACN % ACN 5% ACN % ACN 2% 2 min 9 min 6 min 3 min IPA % IPA 5% IPA % IPA 2% 2 min 9 min 6 min 3 min DMSO % DMSO % DMSO 2% 2 min 9 min 6 min 3 min MeOH % MeOH 5% MeOH % MeOH 2% 2 min 9 min 6 min 3 min TFE % TFE 5% TFE % TFE 2% 3 min 6 min 9 min Figure 4. BSA solubility after digestion with SMART Digest kit. 2 min Tween 2.5% Tween 2.5% Deoxycholate 2 min 9 min 6 min 3 min

5 Conclusion Based on this analysis, the two most effective buffers were combined to create the SMART Digest buffer, which does the following: Enables BSA to be kept in solution, both with and without the addition of trypsin, at concentrations up to mg/ml. Allows higher concentrations of proteins to be kept in solution for longer periods of time, than standard digestion buffers. Application Note 274 Permits use with high-temperature (up to 7 ºC) digestions, enabling digestion and denaturation of proteins to be performed in one step. For Research Use Only thermoscientific.com/smartdigest 25 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. USA and Canada Australia (free call domestic) China (free call domestic) France +33 () Germany +49 () or 2 India AN274-EN 55S Japan (free call domestic) (fax) Korea United Kingdom +44 () New Zealand (free call domestic) Singapore All Other Enquiries +44 () Technical Support For advice and support, please visit our website: